A reversible, H+-transporting ATPase catalyzes ATP synthesis during oxidative phosphorylation. The enzyme is made up of two sectors. F1 lies at the membrane surface, and on isolation catalyzes ATP hydrolysis. F-o extends through the membrane and functions in H+ transport. When the two sectors are properly coupled, ATP synthesis/hydrolysis is coupled to H+- translocation. The F1F-o complexes of mitochondria and Escherichia coli are very similar so general features of one system should be applicable to the other. In E. coli, F1 is composed of 3 pairs of alpha-beta subunits which form catalytic sites, and a single copy of the gamma, delta and epsilon subunits which link F1 to F-o. F-o is composed of 3 subunit types in an a1b2c10 ratio. Subunit c is thought to play a direct role in H+ transport, and in coupling H+ transport to ATP synthesis. It is a protein of 79 residues, folding in the membrane as a hairpin of two alpha-helices. Asp61 (centered in transmembrane helix-2) is thought to mediate H+ transport; H+ transport is thought to alter the loop region of subunit c and initiate a conformational change which ultimately promotes ATP release from alpha-beta subunits in Fl. We have shown that the essential carboxyl of subunit c can be moved from helix-2 to helix-1 and function retained. Analysis of suppressor mutants, optimizing function in the carboxyl-transposition mutant, led to identification of a transmembrane helical surface in subunit a that we now postulate interacts with subunit c. Secondly, polar loop mutants which uncouple H+-transport from ATP synthesis in F1 were characterized. Second site revertants to one of the uncoupled mutants map to a single residue of subunit epsilon. Here, we will better define, by both genetic and physical methods, the interaction between subunits c and a during H+ translocation, and the coupling interaction between the loop of subunit c and subunit epsilon. Additional aims include determination of the solvent accessibility and pKa of Asp6l in situ, and the relationship of this pKa to H+ binding during transport. We have shown that subunit c folds in a chloroform-methanol-H2O solvent much like it is predicted to fold in situ. Further, the unique chemical reactivity of Asp6l is retained. We have derived a low resolution model for the folding of subunit c in this solvent using 2D NMR methods and a novel approach using paramagnetic broadening with a nitroxide derivatived Asp6l. We propose to complete a high resolution solution structure by heteronuclear 3D and 4D methods and to test aspects of the solution model by mutagenesis. This structure will be compared to the structure in detergent micelles, and attempts made to study structural features of c-c and a-c aggregates in an organic solvent mixture. An understanding of structure-function relationships in membrane proteins is fundamental to many problems in biology and medicine. Few intrinsic proteins are understood in any detail. The H+ ATP synthase is central to all cellular functions, i.e. it makes the ATP. Abnormalities in the function of this enzyme, or other mitochondrial respiratory enzymes, result in human disease.